Abstract

Skin wounds need to be repaired rapidly after injury to restore proper skin barrier function. Hydrogen peroxide (H2O2) is a conserved signaling factor that has been shown to promote a variety of skin wound repair processes, including immune cell migration, angiogenesis and sensory axon repair. Despite growing research on H2O2 functions in wound repair, the downstream signaling pathways activated by this reactive oxygen species in the context of injury remain largely unknown. The goal of this study was to provide a comprehensive analysis of gene expression changes in the epidermis upon exposure to H2O2 concentrations known to promote wound repair. Comparative transcriptome analysis using RNA-seq data from larval zebrafish and previously reported microarray data from a human epidermal keratinocyte line shows that H2O2 activates conserved cell migration, adhesion, cytoprotective and anti-apoptotic programs in both zebrafish and human keratinocytes. Further assessment of expression characteristics and signaling pathways revealed the activation of three major H2O2-dependent pathways, EGF, FOXO1, and IKKα. This study expands on our current understanding of the clinical potential of low-level H2O2 for the promotion of epidermal wound repair and provides potential candidates in the treatment of wound healing deficits.

(a) Pools of ~500 larvae/set of 4 day-post-fertilized (dpf) zebrafish larvae were treated with 0.01% (3 mM) H2O2 for three hours and total RNA was subsequently collected followed by pair-end next generation RNA sequencing (n = 3 biological replicates). (b) H2O2 sensor (HPF) either alone or with H2O2 treatment shows that H2O2 is mostly retained in the skin epithelium (n = 5 fish). (c) Distribution of mapped reads in the zebrafish transcriptome. RNA-seq data sent to NCBI (GEO: GSE75728). (d) Transcript complexity between untreated and H2O2-treated larval zebrafish samples. Based on read CPM (counts per million), the left-most value on the X-axis represents the most highly expressed transcripts, which is incrementally summed with each successively lower expressed transcript (rightward). The y-axis (% contribution of the total transcripts) was calculated using: [CPM/sum of all CPM] x 100%. (e) RNA-seq data was normalized with the read CPM method of the number of mapped reads on gene exons. Transcript expression data transformed on M (log ratio of fold change) and A (mean average) scale. Boxed blue regions represent statistically significant transcripts (p < 0.05) returned by the test for differential expression. The MA-plot shows the log2 fold changes from the treatment over the mean of normalized counts, i.e. the average of counts normalized by size factor. Cutoff set to >1 log2 CPM averaged over all samples, and below the cutoff there is no real inferential power. Note: Statistical significance drops below the threshold. (f) Quantitative PCR validation of RNA-seq results of a sub-set of candidate targets. Full data set is presented in Table S7 (n = 3 biological replicates). (g) Heat map indicates unsupervised hierarchical clustering of the top (left) and bottom (right) most significantly enriched transcripts derived from the RNA-seq data after H2O2 treatment. Hierarchical clustering was performed between individual experiments and transcripts. The color key indicates the log2 CPM expression values. Abbreviations: HPF (hydrogen peroxide fluorogenic probe or pentafluorobenzenesulfonyl-fluorescein), CPM: Counts per million, FC: Fold-change.

(a) Downstream Effects Analysis in the Ingenuity’s Pathway Analysis was used to visualize, via color-coded heatmaps, putative biological and disease trends in H2O2-treated zebrafish larvae. Within the cell movement category (boxed in red) are 24 differentially expressed genes (q < 0.1, false discovery rate). The color intensity of the squares in the heatmaps reflects the strength of the absolute z-score for predictions (orange = positive, blue = negative). The categories are assembled with the most significant p-values displayed on the left of the heatmap. The size of the squares reflects the z-score values. (b) Protein classification of transcripts affected by H2O2 treatment of larval zebrafish. Bars represent the % of mapped transcripts to appropriate annotations. Absolute gene numbers are shown in parentheses. (c) Shown is the % of mapped transcripts up (red) or down (blue) regulated for three different categories: oxidoreductase, cell adhesion and ECM. (d) Classification of the molecular functions of affected transcripts after H2O2 treatment. Graphs represent the % of mapped transcripts to appropriate annotations. Analyses were performed using PANTHER.

(a) Causal upstream networks determined using Ingenuity’s Upstream Regulatory Analysis after H2O2 treatment based on the literature compiled in the Ingenuity® Knowledge Base. Fisher’s exact test p-values were calculated to assess the significance of enrichment of the RNA-seq data for the genes downstream of an upstream regulator (Worksheet 4). (b) Most significant downstream genes within the H2O2 upstream network in zebrafish samples (p < 0.05, n = 3 biological replicates). Top numerical value for each transcript represents log2(fold change) (H2O2 vs. untreated), while the lower value represents the p-value. Shades of red indicate the degree of upregulation, while shades of green represent the degree of downregulation. The edges connecting the nodes are colored orange when leading to activation of the downstream node, and yellow if the findings underlying the relationship are inconsistent with the state of the downstream node. Pointed arrowheads indicate that the downstream node is expected to be activated, while blunt arrowheads indicate that the downstream node is expected to be inhibited. (c) Overlap among the differentially expressed genes in H2O2-treated zebrafish and curated chemical-gene interactions for H2O2 derived from the Comparative Toxicogenomics Database (http://ctdbase.org). (d) Functional annotation analysis using DAVID of the H2O2-downstream genes. Enrichment scores ≥1 are considered significant.

Figure 5. Comparison of ARE/EPRE:GFP activation in…

Figure 5. Comparison of ARE/EPRE:GFP activation in zebrafish.

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( a ) The caudal fin of…

Figure 5. Comparison of ARE/EPRE:GFP activation in zebrafish.

(a) The caudal fin of an uninjured EPRE:GFP larval zebrafish was imaged over the course of 12 hours. First and last images of the time-lapse sequence are shown. (b) Matching surface plots and quantification, comparing the fluorescence means of 4 individual fish (n = 4). Statistical significance was tested between first and last time points, showing lack of EPRE:GFP activation by 12 hours. (c) First and last image of a time-lapse sequence showing the amputated caudal fin (arrows) of an EPRE:GFP larval zebrafish. (d) Matching surface plots and quantification, comparing the fluorescence means of 6 individual fish, show that injury fails to activate EPRE:GFP.